(1) Field of the Invention
The present invention relates to a process for purifying insulin and insulin analogs that comprises use of two or more orthogonal chromatographic purification steps in tandem following enzymatic digestion of the propeptide-insulin precursor to remove specific product impurities, improve process consistency, and increase process redundancy in the purification of the insulin or insulin analog, e.g., insulin lispro.
(2) Description of Related Art
Precursor insulin or insulin analogue molecules produced in prokaryote host cells such as E. coli or lower eukaryote host cells such as Saccharomyces cerevisiae or Pichia pastoris are enzymatically cleaved in vitro to remove the connecting peptide joining the B-chain peptide to the A-chain peptide and the N-terminal propeptide to produce insulin or insulin analogue heterodimers. The enzymatic cleavage of precursor insulin or insulin analogue molecules is achieved by digestion with trypsin, carboxypeptidase B, lysC, or combinations thereof. However, the enzymatic digests introduce impurities such as the three amino acid B-chain truncate (des-Thr), deamidoinsulin, arginine-insulin and diarginine-insulin, and insulin ethyl ester.
A key challenge with the purification of insulin (and other insulin analogs) is the downstream removal of product related impurities arising from miscleavage or insufficient cleavage of the propeptide-insulin precursor molecule during the enzymatic digest. Such product impurities include N-terminal propeptide leader and/or signal sequences, C-peptide, dipeptides, aggregated insulin, deamidated insulin, miscleaves, misfolds, and any remaining proinsulin. A significant challenge in the purification of insulin stems from the similarity in chemical structure between insulin and its product-related impurities, which may differ by a single amino acid, requiring selectivity beyond that offered by traditional ion-exchange resins. Additionally, the presence of host cell protein, DNA, and proteolytic enzymes can complicate the purification further by necessitating higher binding capacities and the addition of multiple wash steps.
The following U.S. patents disclose process for purifying proteins or insulins by chromatography: U.S. Pat. Nos. 5,245,008; 4,677,192; 4,129,560; 6,710,167; 5,977,297; and 5,621,073. Karkov et al disclose in J. Chromatography A 1318: 149-155 (2013) a multimodal chromatography method for bind and elute separation of insulin.